Watermelon variety TML-EJ09150

ABSTRACT

The invention provides seed and plants of the watermelon line designated TML-EJ09150. The invention thus relates to the plants, seeds and tissue cultures of watermelon line TML-EJ09150, and to methods for producing a watermelon plant produced by crossing a plant of watermelon line TML-EJ09150 with itself or with another watermelon plant, such as a plant of another line. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of a plant of watermelon line TML-EJ09150, including the fruits and gametes of such plants.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and, morespecifically, to the development of watermelon line TML-EJ09150.

BACKGROUND OF THE INVENTION

The goal of vegetable breeding is to combine various desirable traits ina single variety/hybrid. Such desirable traits may include any traitdeemed beneficial by a grower and/or consumer, including greater yield,resistance to insects or disease, tolerance to environmental stress, andnutritional value.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant or plant variety. A plant cross-pollinates if pollencomes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants of different genotypes produces auniform population of hybrid plants that are heterozygous for many geneloci. Conversely, a cross of two plants each heterozygous at a number ofloci produces a population of hybrid plants that differ genetically andare not uniform. The resulting non-uniformity makes performanceunpredictable.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines are developed byselfing and selection of desired phenotypes. The new lines are evaluatedto determine which of those have commercial potential.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a watermelon plant of thewatermelon line designated TML-EJ09150. Also provided are watermelonplants having all the physiological and morphological characteristics ofwatermelon line TML-EJ09150. Parts of the watermelon plant of thepresent invention are also provided, for example, including pollen, anovule, scion, a rootstock, a fruit, and a cell of the plant.

The invention also concerns seed of watermelon line TML-EJ09150. Thewatermelon seed of the invention may be provided, in certainillustrative embodiments, as an essentially homogeneous population ofwatermelon seed of the line designated TML-EJ09150. Essentiallyhomogeneous populations of seed are generally free from substantialnumbers of other seed. Therefore, in one embodiment, seed of lineTML-EJ09150 may be defined as forming at least about 90% of the totalseed, including at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more of the seed. The population of watermelon seed may beparticularly defined as being essentially free from hybrid seed. Theseed population may be separately grown to provide an essentiallyhomogeneous population of watermelon plants designated TML-EJ09150.

In another aspect of the invention, a plant of watermelon lineTML-EJ09150 comprising an added heritable trait is provided. Theheritable trait may comprise a genetic locus that is, for example, adominant or recessive allele. In one embodiment of the invention, aplant of watermelon line TML-EJ09150 is defined as comprising a singlelocus conversion. In specific embodiments of the invention, an addedgenetic locus confers one or more traits such as, for example, herbicidetolerance, insect resistance, disease resistance, and modifiedcarbohydrate metabolism. In further embodiments, the trait may beconferred by a naturally occurring gene introduced into the genome ofthe line by backcrossing, a natural or induced mutation, or a transgeneintroduced through genetic transformation techniques into the plant or aprogenitor of any previous generation thereof. When introduced throughtransformation, a genetic locus may comprise one or more genesintegrated at a single chromosomal location.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of line TML-EJ09150 is provided. The tissue culturewill preferably be capable of regenerating plants capable of expressingall of the physiological and morphological characteristics of the line,and of regenerating plants having substantially the same genotype asother plants of the line. Examples of some of the physiological andmorphological characteristics of the line TML-EJ09150 include thosetraits set forth in the tables herein. The regenerable cells in suchtissue cultures may be derived, for example, from embryos, meristems,cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower,seed and stalks. Still further, the present invention provideswatermelon plants regenerated from a tissue culture of the invention,the plants having all the physiological and morphologicalcharacteristics of line TML-EJ09150.

In yet another aspect of the invention, processes are provided forproducing watermelon seeds, plants and fruits, which processes generallycomprise crossing a first parent watermelon plant with a second parentwatermelon plant, wherein at least one of the first or second parentwatermelon plants is a plant of the line designated TML-EJ09150. Theseprocesses may be further exemplified as processes for preparing hybridwatermelon seed or plants, wherein a first watermelon plant is crossedwith a second watermelon plant of a different, distinct line to providea hybrid that has, as one of its parents, the watermelon plant lineTML-EJ09150. In these processes, crossing will result in the productionof seed. The seed production occurs regardless of whether the seed iscollected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and second parent watermelon plant,often in proximity so that pollination will occur for example, mediatedby insect vectors. Alternatively, pollen can be transferred manually.Where the plant is self-pollinated, pollination may occur without theneed for direct human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first andsecond parent watermelon plants into plants that bear flowers. A thirdstep may comprise preventing self-pollination of the plants, such as byemasculating the flowers, (i.e., killing or removing pollen).

A fourth step for a hybrid cross may comprise cross-pollination betweenthe first and second parent watermelon plants. Yet another stepcomprises harvesting the seeds from at least one of the parentwatermelon plants. The harvested seed can be grown to produce awatermelon plant or hybrid watermelon plant.

The present invention also provides the watermelon seeds and plantsproduced by a process that comprises crossing a first parent watermelonplant with a second parent watermelon plant, wherein at least one of thefirst or second parent watermelon plants is a plant of the linedesignated TML-EJ09150. In one embodiment of the invention, watermelonseed and plants produced by the process are first generation (F1) hybridwatermelon seed and plants produced by crossing a plant in accordancewith the invention with another, distinct plant. The present inventionfurther contemplates plant parts of such an F1 hybrid watermelon plant,and methods of use thereof. Therefore, certain exemplary embodiments ofthe invention provide an F1 hybrid watermelon plant and seed thereof.

In still yet another aspect of the invention, the genetic complement ofthe watermelon plant line designated TML-EJ09150 is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a watermelon plant, or a cell or tissue of thatplant. A genetic complement thus represents the genetic makeup of acell, tissue or plant, and a hybrid genetic complement represents thegenetic make up of a hybrid cell, tissue or plant. The invention thusprovides watermelon plant cells that have a genetic complement inaccordance with the watermelon plant cells disclosed herein, and plants,seeds and plants containing such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that line TML-EJ09150 could be identified by any of themany well known techniques such as, for example, Simple Sequence LengthPolymorphisms (SSLPs) (Williams et al., Nucleic Acids Res., 18:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by watermelon plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a watermelon plant of the invention with a haploid geneticcomplement of a second watermelon plant, preferably, another, distinctwatermelon plant. In another aspect, the present invention provides awatermelon plant regenerated from a tissue culture that comprises ahybrid genetic complement of this invention.

In still yet another aspect, the invention provides a method ofdetermining the genotype of a plant of watermelon line TML-EJ09150comprising detecting in the genome of the plant at least a firstpolymorphism. The method may, in certain embodiments, comprise detectinga plurality of polymorphisms in the genome of the plant. The method mayfurther comprise storing the results of the step of detecting theplurality of polymorphisms on a computer readable medium. The inventionfurther provides a computer readable medium produced by such a method.

In still yet another aspect, the present invention provides a method ofproducing a plant derived from line TML-EJ09150, the method comprisingthe steps of: (a) preparing a progeny plant derived from lineTML-EJ09150, wherein said preparing comprises crossing a plant of theline TML-EJ09150 with a second plant; and (b) crossing the progeny plantwith itself or a second plant to produce a seed of a progeny plant of asubsequent generation. In further embodiments, the method mayadditionally comprise: (c) growing a progeny plant of a subsequentgeneration from said seed of a progeny plant of a subsequent generationand crossing the progeny plant of a subsequent generation with itself ora second plant; and repeating the steps for an additional 3-10generations to produce a plant derived from line TML-EJ09150. The plantderived from line TML-EJ09150 may be an inbred line, and theaforementioned repeated crossing steps may be defined as comprisingsufficient inbreeding to produce the inbred line. In the method, it maybe desirable to select particular plants resulting from step (c) forcontinued crossing according to steps (b) and (c). By selecting plantshaving one or more desirable traits, a plant derived from lineTML-EJ09150 is obtained which possesses some of the desirable traits ofthe line as well as potentially other selected traits.

In certain embodiments, the present invention provides a method ofproducing watermelon comprising: (a) obtaining a plant of watermelonline TML-EJ09150, wherein the plant has been cultivated to maturity, and(b) collecting watermelons from the plant.

These and other features and advantages of this invention are describedin, or are apparent from, the following detailed description of variousexemplary embodiments of the devices and methods according to thisinvention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of the watermelon line designated TML-EJ09150.This line shows uniformity and stability within the limits ofenvironmental influence for the traits described hereinafter. watermelonline TML-EJ09150 provides sufficient seed yield. By crossing with adistinct second plant, uniform F1 hybrid progeny can be obtained.

Watermelon line TML-EJ09150 is a tetraploid watermelon inbred with amedium seed size that produces light green fruits with no stripes. It isbelieved to be similar to variety Mickylee, but is tetraploid.

A. Origin and Breeding History of TML-EJ09150

The crossing and selections that led directly to TML-EJ09150 can besummarized as follows:

Line TML-EJ09150 was obtained by pedigree breeding through crossingparental lines TCS-110-1009, female parent used commercially in triploidhybrid Olympia, and Appletexture. Both parental lines had been developedby Barham Seeds, which was acquired by Seminis Seeds in 1999, which wasacquired by Monsanto in 2005. Line TCS-110-1009 is a tetraploid inbredline with Crimson sweet-type fruits. Appletexture is a firm-fleshtetraploid inbred with Mickylee type fruits, but that has not been usedcommercially.

The original cross was done in Tifton Ga. in spring of Year 1. F1 plantswere self-pollinated in Salama, Guatemala in the fall of Year 1. F2plants were self-pollinated in Woodland, Calif. in the summer of Year 2.F3 plants were self-pollinated in the spring of Year 3 in Felda, Fla. F4plants were self-pollinated in the summer of Year 4. F5 plants wereself-pollinated in Felda, Fla. in the spring of Year 5. F6 plants wereself-pollinated in Felda, Fla. in the fall of Year 5. F7 plants wereself-pollinated in Felda, Fla. in the spring of Year 6. F8 generationwas grown in Salama, Guatemala, in the fall of Year 6. F9 generation wasgrown in Woodland, Calif. in summer Year 7. F10 generation was grown ina greenhouse in Woodland, Calif. during the spring of Year 10 and bulkharvested. The F11 seeds were provided to our Foundation Seed departmentand increased in a cage in the summer of Year 10. At each generationselection was made for fruits with a high flesh firmness and good seednumbers.

TML-EJ09150 is uniform and stable. A small percentage of variants canoccur within commercially acceptable limits for almost anycharacteristic during the course of repeated multiplication. However novariants are expected.

B. Physiological and Morphological Characteristics of Watermelon LineTML-EJ09150

In accordance with one aspect of the present invention, there isprovided a plant having the physiological and morphologicalcharacteristics of watermelon line TML-EJ09150. A description of thephysiological and morphological characteristics of watermelon lineTML-EJ09150 is presented in Table 1.

TABLE 1 Physiological and Morphological Characteristics of LineTML-EJ09150 Comparison Variety- CHARACTERISTIC TML-EJ09150TML-110-1434 1. Type round round 2. Maturity days relative maturity (asreported 116 116 in seed catalogs) category late late 3. Ploidytetraploid tetraploid 4. Plant sex form monoecious monoecious cotyledonshape flat flat cotyledon: size small small cotyledon: shape narrowelliptic medium elliptic cotyledon: intensity of green color mediummedium number of main stems at crown 4.1 3.8 number of staminate flowersper 48.2 49.2 plant at first fruit set number of pistillate flowers per3.7 3.5 plant at first fruit set number of perfect flowers per plant 0 0at first fruit set 5. Stem shape in cross-section round round diameterat second node 12.4 mm 9.1 mm surface pubescent pubescent cm vine length(at last harvest) 272.4 cm 268.6 cm number of internodes (at last 40.637 harvest) ratio: cm vine length ÷ number of 6.6 6.8 internodes (atlast harvest) 6. Leaf shape ovate ovate lobes lobed lobed length 13.6 cm14.9 cm width 15.8 cm 17.7 cm size ratio wider than long wider than longleaf blade: size medium medium leaf blade: ratio length/width mediummedium dorsal surface pubescence pubescent pubescent ventral surfacepubescence pubescent pubescent color gray-green gray-green RHS colorchart value for the leaf 189A 189A color leaf blade: color greyish greengray-green leaf blade: color of veins green green leaf blade: degree oflobing medium strong leaf blade: blistering weak medium 7. Flowerdiameter across the staminate 3.4 cm 3.2 cm flower diameter across thepistillate flower 3.0 cm 2.8 cm diameter across the perfect flower 0 cm0 cm color yellow yellow RHS color chart value for flower  4B  8B color8. Mature Fruit shape round round fruit: shape in longitudinal sectioncircular circular depression at base shallow shallow fruit: shape ofapical part truncate rounded fruit: depression at apex shallow shallowlength 24.2 cm 21.5 cm diameter at midsection 23.5 cm 21.3 cm fruit:weight medium low to medium average weight 6.7 kg 5.0 kg maximum weight11.7 kg 8.6 kg index = length ÷ diameter × 10 10.2 10 surface smoothsmooth skin color pattern solid (one color) stripe primary skin colorlight green light green (Charleston Grey) RHS color chart value forprimary 145D 145C skin color secondary skin color medium green mediumgreen RHS color chart value for 137A 137C secondary skin color fruit:ground color* of skin very light green very light to light *Ground coloris the lightest color green of the skin. In striped fruit, the darkercolor of the skin concerns the stripes. fruit: conspicuousness ofveining medium inconspicuous or very weakly conspicuous fruit: patternof stripes only veins only veins fruit: width of stripes very narrowvery narrow fruit: main color of stripes medium green medium greenfruit: conspicuousness of stripes inconspicuous or very inconspicuous orvery weakly conspicuous weakly conspicuous fruit: margin of stripesdiffuse diffuse fruit: size of insertion of peduncle small small fruit:size of pistil scar medium medium fruit: grooving absent or very weakabsent or very weak fruit: waxy layer absent or very weak absent or veryweak 9. Rind texture tough tough thickness of blossom end 11.9 mm 10.6mm thickness of sides 14.8 mm 12.6 mm fruit: thickness of pericarp thickmedium 10. Flesh texture crisp crisp coarseness fine-little fiberfine-little fiber fruit: main color of flesh red red RHS color chartvalue for the main  39A  34B flesh color of mature fruit refractometer %soluble solids of 11.80% 9.90% juice (center of fruit) % hollow heart   0%   0% % placental separation   10%   10% % transverse crack    0%  0% only diploid and tetraploid none or few none or few varieties:fruit: number of seeds only diploid and tetraploid medium mediumvarieties: seed: length only diploid and tetraploid low low varieties:seed: ratio length/width only diploid and tetraploid brown brownvarieties: seed: ground color of testa only diploid and tetraploidabsent present varieties: seed: over color of testa only diploid andtetraploid small very large varieties: seed: area over color in relationto that of ground color only diploid and tetraploid medium mediumvarieties: seed: patches in hilum 11. Seed size medium medium length 9.7mm 9.7 mm width 6.6 mm 6.5 mm thickness 2.4 mm 2.6 mm index 14.7 14.8(index = length ÷ diameter × 10) grams per 1000 seeds 50.0 g 73.0 gmnumber of seeds per fruit 48.8 82.2 color dark brown dark brown RHScolor chart value for the seed 166B 164C color *These are typicalvalues. Values may vary due to environment. Other values that aresubstantially equivalent are within the scope of the invention.

C. Breeding Watermelon Line TML-EJ09150

One aspect of the current invention concerns methods for crossing thewatermelon line TML-EJ09150 with itself or a second plant and the seedsand plants produced by such methods. These methods can be used forpropagation of line TML-EJ09150, or can be used to produce hybridwatermelon seeds and the plants grown therefrom. Hybrid seeds areproduced by crossing line TML-EJ09150 with second watermelon parentline.

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing line TML-EJ09150 followed by multiplegenerations of breeding according to such well known methods. Newvarieties may be created by crossing with any second plant. In selectingsuch a second plant to cross for the purpose of developing novel lines,it may be desired to choose those plants which either themselves exhibitone or more selected desirable characteristics or which exhibit thedesired characteristic(s) in progeny. Once initial crosses have beenmade, inbreeding and selection take place to produce new varieties. Fordevelopment of a uniform line, often five or more generations of selfingand selection are involved.

Uniform lines of new varieties may also be developed by way ofdouble-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner, true breeding lines can be produced in aslittle as one generation. Haploid embryos may be produced frommicrospores, pollen, anther cultures, or ovary cultures. The haploidembryos may then be doubled autonomously, or by chemical treatments(e.g. colchicine treatment). Alternatively, haploid embryos may be growninto haploid plants and treated to induce chromosome doubling. In eithercase, fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with lineTML-EJ09150 and progeny thereof to achieve a homozygous line.

New varieties may be created, for example, by crossing line TML-EJ09150with any second plant and selection of progeny in various generationsand/or by doubled haploid technology. In choosing a second plant tocross for the purpose of developing novel lines, it may be desired tochoose those plants which either themselves exhibit one or more selecteddesirable characteristics or which exhibit the desired characteristic(s)in progeny. After one or more lines are crossed, true-breeding lines maybe developed.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred.

The line of the present invention is particularly well suited for thedevelopment of new lines based on the elite nature of the geneticbackground of the line. In selecting a second plant to cross withTML-EJ09150 for the purpose of developing novel watermelon lines, itwill typically be preferred to choose those plants which eitherthemselves exhibit one or more selected desirable characteristics orwhich exhibit the desired characteristic(s) when in hybrid combination.Examples of desirable characteristics may include, in specificembodiments, high seed yield, high seed germination, seedling vigor,high fruit yield, disease tolerance or resistance, and adaptability forsoil and climate conditions. Consumer-driven traits, such as fruitshape, color, texture, and taste are other examples of traits that maybe incorporated into new lines of watermelon plants developed by thisinvention.

D. Further Embodiments of the Invention

In certain aspects of the invention, plants described herein areprovided modified to include at least a first desired heritable trait.Such plants may, in one embodiment, be developed by a plant breedingtechnique called backcrossing, wherein essentially all of thephysiological and morphological characteristics of a variety arerecovered in addition to a genetic locus transferred into the plant viathe backcrossing technique. The term single locus converted plant asused herein refers to those watermelon plants which are developed by aplant breeding technique called backcrossing, wherein essentially all ofthe desired physiological and morphological characteristics of a varietyare recovered in addition to the single locus transferred into thevariety via the backcrossing technique.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the present variety. The parentalwatermelon plant which contributes the locus for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental watermelon plant to which the locus or loci from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a watermelon plant isobtained wherein essentially all of the desired physiological andmorphological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred locus fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered and the genetic distance between the recurrentand nonrecurrent parents. Although backcrossing methods are simplifiedwhen the characteristic being transferred is a dominant allele, arecessive allele, or an additive allele (between recessive anddominant), may also be transferred. In this instance it may be necessaryto introduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

In one embodiment, progeny watermelon plants of a backcross in whichTML-EJ09150 is the recurrent parent comprise (i) the desired trait fromthe non-recurrent parent and (ii) all of the physiological andmorphological characteristics of watermelon line TML-EJ09150 asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

Watermelon varieties can also be developed from more than two parents.The technique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

With the development of molecular markers associated with particulartraits, it is possible to add additional traits into an established germline, such as represented here, with the end result being substantiallythe same base germplasm with the addition of a new trait or traits.Molecular breeding, as described in Moose and Mumm, 2008 (PlantPhysiology, 147: 969-977), for example, and elsewhere, provides amechanism for integrating single or multiple traits or QTL into an eliteline. This molecular breeding-facilitated movement of a trait or traitsinto an elite line may encompass incorporation of a particular genomicfragment associated with a particular trait of interest into the eliteline by the mechanism of identification of the integrated genomicfragment with the use of flanking or associated marker assays. In theembodiment represented here, one, two, three or four genomic loci, forexample, may be integrated into an elite line via this methodology. Whenthis elite line containing the additional loci is further crossed withanother parental elite line to produce hybrid offspring, it is possibleto then incorporate at least eight separate additional loci into thehybrid. These additional loci may confer, for example, such traits as adisease resistance or a fruit quality trait. In one embodiment, eachlocus may confer a separate trait. In another embodiment, loci may needto be homozygous and exist in each parent line to confer a trait in thehybrid. In yet another embodiment, multiple loci may be combined toconfer a single robust phenotype of a desired trait.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,modified fatty acid or carbohydrate metabolism, and altered nutritionalquality. These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. For this selection process, the progeny of the initialcross are assayed for viral resistance and/or the presence of thecorresponding gene prior to the backcrossing. The selection eliminatesany plants that do not have the desired gene and resistance trait, andonly those plants that have the trait are used in the subsequentbackcross. This process is then repeated for all additional backcrossgenerations.

Selection of watermelon plants for breeding is not necessarily dependenton the phenotype of a plant and instead can be based on geneticinvestigations. For example, one can utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers can be used to identify the presence or absence of a trait inthe offspring of a particular cross, and can be used in selection ofprogeny for continued breeding. This technique is commonly referred toas marker assisted selection. Any other type of genetic marker or otherassay which is able to identify the relative presence or absence of atrait of interest in a plant can also be useful for breeding purposes.Procedures for marker assisted selection applicable to the breeding ofwatermelon are well known in the art. Such methods will be of particularutility in the case of recessive traits and variable phenotypes, orwhere conventional assays may be more expensive, time consuming orotherwise disadvantageous. Types of genetic markers which could be usedin accordance with the invention include, but are not necessarilylimited to, Simple Sequence Length Polymorphisms (SSLPs) (Williams etal., Nucleic Acids Res., 1 8:6531-6535, 1990), Randomly AmplifiedPolymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),Sequence Characterized Amplified Regions (SCARs), Arbitrary PrimedPolymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (EP 534 858, specifically incorporated herein byreference in its entirety), and Single Nucleotide Polymorphisms (SNPs)(Wang et al., Science, 280:1077-1082, 1998).

E. Plants Derived From Watermelon Line TML-EJ09150 by GeneticEngineering

Many useful traits that can be introduced by backcrossing, as well asdirectly into a plant, are those which are introduced by genetictransformation techniques. Genetic transformation may therefore be usedto insert a selected transgene into the watermelon line of the inventionor may, alternatively, be used for the preparation of transgenes whichcan be introduced by backcrossing. Methods for the transformation ofplants that are well known to those of skill in the art and applicableto many crop species include, but are not limited to, electroporation,microprojectile bombardment, Agrobacterium-mediated transformation anddirect DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

An efficient method for delivering transforming DNA segments to plantcells is microprojectile bombardment. In this method, particles arecoated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target watermelon cells. The screen disperses the particlesso that they are not delivered to the recipient cells in largeaggregates. Microprojectile bombardment techniques are widelyapplicable, and may be used to transform virtually any plant species.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., Bio-Technology, 3(7):637-642, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes. Additionally, Agrobacterium containing both armed anddisarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Bio/Technology, 3:629-635, 1985; U.S.Pat. No. 5,563,055).

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al.,Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature,312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986;Marcotte et al., Nature, 335:454, 1988). Transformation of plants andexpression of foreign genetic elements is exemplified in Choi et al.(Plant Cell Rep., 13: 344-348, 1994), and Ellul et al. (Theor. Appl.Genet., 107:462-469, 2003).

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for plant gene expressioninclude, but are not limited to, the cauliflower mosaic virus (CaMV)P-35S promoter, which confers constitutive, high-level expression inmost plant tissues (see, e.g., Odel et al., Nature, 313:810, 1985),including in monocots (see, e.g., Dekeyser et al., Plant Cell, 2:591,1990; Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990); a tandemlyduplicated version of the CaMV 35S promoter, the enhanced 35S promoter(P-e35S) the nopaline synthase promoter (An et al., Plant Physiol.,88:547, 1988), the octopine synthase promoter (Fromm et al., Plant Cell,1:977, 1989); and the figwort mosaic virus (P-FMV) promoter as describedin U.S. Pat. No. 5,378,619 and an enhanced version of the FMV promoter(P-eFMV) where the promoter sequence of P-FMV is duplicated in tandem,the cauliflower mosaic virus 19S promoter, a sugarcane bacilliform viruspromoter, a commelina yellow mottle virus promoter, and other plant DNAvirus promoters known to express in plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals can alsobe used for expression of an operably linked gene in plant cells,including promoters regulated by (1) heat (Callis et al., PlantPhysiol., 88:965, 1988), (2) light (e.g., pea rbcS-3A promoter,Kuhlemeier et al., Plant Cell, 1:471, 1989; maize rbcS promoter,Schaffner and Sheen, Plant Cell, 3:997, 1991; or chlorophyll a/b-bindingprotein promoter, Simpson et al., EMBO J., 4:2723, 1985), (3) hormones,such as abscisic acid (Marcotte et al., Plant Cell, 1:969, 1989), (4)wounding (e.g., wunl, Siebertz et al., Plant Cell, 1:961, 1989); or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters (e.g., Roshal etal., EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988;Bustos et al., Plant Cell, 1:839, 1989).

Exemplary nucleic acids which may be introduced to plants of thisinvention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a watermelon plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a watermelon plant includeone or more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference in their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillito, Mol.Biotech., 7:125, 1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

F. Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration: The development of a plant from tissue culture.

Resistance: As used herein, the terms “resistance” and “tolerance” areused interchangeably to describe plants that show no symptoms to aspecified biotic pest, pathogen, abiotic influence or environmentalcondition. These terms are also used to describe plants showing somesymptoms but that are still able to produce marketable product with anacceptable yield. Some plants that are referred to as resistant ortolerant are only so in the sense that they may still produce a crop,even though the plants are stunted and the yield is reduced.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing wherein essentially allof the physiological and morphological characteristics of an inbred arerecovered in addition to the characteristics conferred by the singlelocus transferred into the inbred via the backcrossing technique. By“essentially all,” it is meant that all of the characteristics of aplant are recovered that are otherwise present when compared in the sameenvironment and save for the converted locus, other than an occasionalvariant trait that might arise during backcrossing or directintroduction of a transgene. A single locus may comprise one gene, or inthe case of transgenic plants, one or more transgenes integrated intothe host genome at a single site (locus).

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tetraploid: A cell or organism having four sets of chromosomes.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a garden watermelon plant bytransformation.

G. Deposit Information

A deposit of watermelon line TML-EJ09150, disclosed above and recited inthe claims, has been made with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, USA, andassigned ATCC Accession No. PTA-121212. The seeds were deposited withthe ATCC on May 10, 2014. Access to this deposit will be availableduring the pendency of the application to the Commissioner of Patentsand Trademarks and persons determined by the Commissioner to be entitledthereto upon request. The deposits will be maintained in the ATCCDepository, which is a public depository, for a period of 30 years, or 5years after the most recent request, or for the enforceable life of thepatent, whichever is longer, and will be replaced if it becomesnonviable during that period. Applicant does not waive rights grantedunder this patent or under the Plant Variety Protection Act (7 U.S.C.2321 et seq.).

Although the foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the invention, as limited only by the scope of theappended claims.

All references cited herein are hereby expressly incorporated herein byreference.

What is claimed is:
 1. A seed of watermelon line TML-EJ09150, a sampleof seed of said line having been deposited under ATCC Accession NumberPTA-121212.
 2. A plant of watermelon line TML-EJ09150, a sample of seedof said line having been deposited under ATCC Accession NumberPTA-121212.
 3. A plant part of the plant of claim
 2. 4. The plant partof claim 3, wherein said part is selected from the group consisting of apollen, an ovule, scion, a rootstock, a fruit, and a cell of the plant.5. A watermelon plant, or a part thereof, having all the physiologicaland morphological characteristics of the watermelon plant of claim
 2. 6.A tissue culture of regenerable cells of watermelon line TML-EJ09150, asample of seed of said line having been deposited under ATCC AccessionNumber PTA-121212.
 7. The tissue culture according to claim 6,comprising cells or protoplasts from a plant part selected from thegroup consisting of embryos, meristems, cotyledons, pollen, leaves,anthers, roots, root tips, pistil, flower, seed and stalks.
 8. Awatermelon plant regenerated from the tissue culture of claim 6, whereinthe regenerated plant expresses all of the physiological andmorphological characteristics of watermelon line TML-EJ09150, a sampleof seed of said line having been deposited under ATCC Accession NumberPTA-121212.
 9. A method of producing watermelon seed, comprisingcrossing the plant of claim 2 with itself or a second watermelon plant.10. The method of claim 9, wherein the plant of watermelon lineTML-EJ09150 is the female parent.
 11. The method of claim 9, wherein theplant of watermelon line TML-EJ09150 is the male parent.
 12. An F1hybrid seed produced by the method of claim
 9. 13. An F1 hybrid plantproduced by growing the seed of claim
 12. 14. A method for producing aseed of a line TML-EJ09150-derived watermelon plant comprising the stepsof: (a) crossing a watermelon plant of line TML-EJ09150 with a secondwatermelon plant, a sample of seed of said line having been depositedunder ATCC Accession Number PTA-121212; and (b) allowing seed of aTML-EJ09150-derived watermelon plant to form.
 15. The method of claim14, further comprising the steps of: (c) crossing a plant grown fromsaid TML-EJ09150-derived watermelon seed with itself or a secondwatermelon plant to yield additional TML-EJ09150-derived watermelonseed; (d) growing said additional TML-EJ09150-derived watermelon seed ofstep (c) to yield additional TML-EJ09150-derived watermelon plants; and(e) repeating the crossing and growing steps of (c) and (d) to generatefurther TML-EJ09150-derived watermelon plants.
 16. A method ofvegetatively propagating a plant of watermelon line TML-EJ09150comprising the steps of: (a) collecting tissue capable of beingpropagated from a plant of watermelon line TML-EJ09150, a sample of seedof said line having been deposited under ATCC Accession NumberPTA-121212; (b) cultivating said tissue to obtain proliferated shoots;and (c) rooting said proliferated shoots to obtain rooted plantlets. 17.The method of claim 16, further comprising growing plants from saidrooted plantlets.
 18. A method of introducing a desired trait into awatermelon plant, said method comprising: (a) crossing a plant of lineTML-EJ09150 with a second watermelon plant that comprises a desiredtrait to produce F1 progeny, a sample of seed of said line TML-EJ09150having been deposited under ATCC Accession Number PTA-121212; (b)selecting an F1 progeny that comprises the desired trait; (c) crossingthe selected F1 progeny with a plant of line TML-EJ09150 to producebackcross progeny; (d) selecting backcross progeny comprising thedesired trait and the physiological and morphological characteristics ofwatermelon line TML-EJ09150; and (e) repeating steps (c) and (d) threeor more times to produce selected fourth or higher backcross progenythat comprise the desired trait.
 19. A watermelon plant produced by themethod of claim
 18. 20. A method of producing a plant of watermelon lineTML-EJ09150 comprising an added desired trait, the method comprisingintroducing a transgene conferring the desired trait into a plant ofwatermelon line TML-EJ09150, a sample of seed of said line TML-EJ09150having been deposited under ATCC Accession Number PTA-121212.
 21. A seedof the plant of claim
 19. 22. A method of producing watermelonscomprising: (a) obtaining the plant of claim 2, wherein the plant hasbeen cultivated to maturity, and (b) collecting at least one watermelonfrom the plant.